Cytomegalovirus drives Vδ1+ γδ T cell expansion and clonality in common variable immunodeficiency

The function and phenotype of γδ T cells in the context of common variable immunodeficiency (CVID) has not been explored. CVID is a primary immunodeficiency disorder characterized by impaired antibody responses resulting in increased susceptibility to infections. γδ T cells are a subset of unconventional T cells that play crucial roles in host defence against infections. In this study, we aim to determine the roles and functions of γδ T cells in CVID. We observe a higher frequency of Vδ1+ γδ T cells compared to healthy controls, particularly in older patients. We also find a higher proportion of effector-memory Vδ1+ γδ T cells and a more clonal T cell receptor (TCR) repertoire in CVID. The most significant driver of the Vδ1+ γδ T cell expansion and phenotype in CVID patients is persistent cytomegalovirus (CMV) viremia. These findings provide valuable insights into γδ T cell biology and their contribution to immune defence in CVID.

The study by Chan et al. investigated in γδ T cells in the context of CVID disease, a disease characterized by impaired B cell responses.Studying γδ T cells in immunodeficient patients provides an excellent tool to elucidate on the biology of human γδ T cells.From this perspective results of this study provide valuable data.Nevertheless, as it has been reported by many other groups it is well established that Vd1 T cells respond to CMV infection/reactivation in immunocompromised patients.The main findings of this study are that γδ T cells are not impacted in CVID, and are capable to response to CMV viremia.I have following comments on the results: 1.The authors focused on characterizing γδ T cells.To place findings in a general context, I recommend to also show results, namely FACS data, for CD4 and CD8 αβ T cells.At least in the methods it is stated that anti-CD4 and anti-Va7.2antibodies were included, and frequencies of CD4 and CD8 γδ T cells were examined.The analysis of CD8+ αβ T cells is of particular interest, when studying CMV viremia.Have the authors also performed TCR-seq of ab T cells as it would be important to also elucidate on clonal expansion of ab T cells upon CMV? 2. The authors claim that age and CMV impact on γδ T cell subset distribution in CVID, but only looked at various clinical parameters (gender, CMV, age, disease) separately (Fig. 2 -3, maybe also Fig. 4).It is important to perform a statistical analysis on the abundance of Vδ1 and Vδ2 T cells using a multivariate linear regression model, and examine the ß-estimates for the respective parameters.This paper describes phenotypic and quantitative characteristics of γδ T cells in the context of common variable immunodeficiency (CVID), which is a heterogeneous disease with low antibody levels.For some analyses, CVID patients were stratified by cytomegalo virus (CMV) viremia.One question that was raised was whether normal γδ T cell function requires intact B cell immunity, which is an interesting question given the presence of Fc receptors on gd T cells.

Does the phenotype of γδ T cells change with age in
The main findings were that CVID and CMV are positively correlated with Vd1 expansion and cytotoxic phenotype.For CVID, this is a new finding.However, it has been described in several prominent papers that are cited in the current paper, that Vd1 cells expand in response to CMV and show increased cytotoxic phenotype, so this aspect of the findings is not novel.The main conclusion of the current paper is that the γδ T cell population is not limited in size or changed in phenotype due to impaired B cell immunity in CVID.
All experiments, including flow cytometry, are technically well performed, analyzed, and clearly reported.The patient cohort is well described.My enthusiasm for this work is limited because it is mostly descriptive and correlative and does not lead to new disease-related hypotheses or new insights into the function of gd T cells.For this reason, I do not consider this work of significance to the general field of immunology or even the narrow field of gd T cell biology.This is an extremely well written manuscript that describes the gdT cell profile in 5 patients with CMV viraemia and CVID.The work also describes findings in a wide range of healthy donors and CVID cohort.
The findings are complementary to many studies of gdT cells in CMV disease in other settings but are new in this situation.The fact that 4 of the 5 patients had an NFKB mutation is noteworthy (although not overinterpreted) The findings show expansion of private clones of Vd1 T cells with CMV viraemia.The specificity of these is unknown.
The writing and presentation is very clear.I would suggest some discussion of number vs percentage.As I see it, the number of Vd1 T cells in CMV CVID is not increased (this is put in supplementary -perhaps should be main figure).Perhaps surprising given the article?But the % is increased over healthy CMV-donors (not a great control).And the % of Vd1 increases.So does this mean there is lymphopenia in CMV CVID and the retained Vd1 pool starts to represent a higher % ?I would suggest rephrasing of the statement "Our results suggest that any future treatments for CMV that specifically target Vδ1+ γδ T cells would likely be effective in CVID patients".Does this mean kill them off or enhance them?Also, if they are there and expanded, why is disease still there?Are the populations exhausted?Indeed, it was not clear if the functional properties of the Vd1 cells in CMV CVID was determined.

We are very pleased Reviewer 1 states: "Studying γδ T cells in immunodeficient patients provides an excellent tool to elucidate on the biology of human γδ T cells. From this perspective results of this study provide valuable data".
Comment 1:

The authors focused on characterizing γδ T cells. To place findings in a general context, I recommend to also show results, namely FACS data, for CD4 and CD8 αβ T cells. At least in the methods it is stated that anti-CD4 and anti-Va7.2 antibodies were included, and frequencies of CD4 and CD8 γδ T cells were examined. The analysis of CD8+ αβ T cells is of particular interest, when studying CMV viremia. Have the authors also performed TCR-seq of ab T cells as it would be important to also elucidate on clonal expansion of ab T cells upon CMV?
Response: We agree with Reviewer 1 that the inclusion of FACS data on the CD4 and CD8 T cell frequencies will provide important contextual insight into the results of the paper.Therefore, we have included the circulating absolute numbers of: total T cells as well as conventional CD4 and CD8 T cells (with the normal healthy range indicated) in Supplementary Fig. 7b, frequencies for each CVID patient can be found in Supplementary Table 2 and we have added the following to the main text: (page 7, line 157-159) "As all other T cell counts were not significantly different across cohorts and largely within the normal range (Supplementary Fig. 7b) this suggests the expansion of the Vδ1 + subset is at the expense of the Vδ2 + subset."

Response:
This has now been updated with the Bioproject accession number: PRJNA1055292 (page 18, line 441-442).To improve clarity, Fig. 1b, 4b, and Supplementary Fig. 5b log scales have all been converted to linear.The absolute counts for γδ T cell subsets have also been included in Supplementary Fig. 2a and added the following to the main text: (page 5, line 102-104) "This resulted in a significantly higher Vδ1/Vδ2 ratio in CVID (Fig. 1c), however, the cell counts for the γδ subsets were not significantly different between healthy and CVID (Supplementary Fig. 2a)."

Response:
We have now updated all figure legends to include exact n values for each graph (rather than a range given for all graphs in figure).

Reviewer #2
We are pleased Reviewer 2 states: "All experiments, including flow cytometry, are technically well performed, analyzed, and clearly reported.The patient cohort is well described."

Comment 1
My enthusiasm for this work is limited because it is mostly descriptive and correlative and does not lead to new disease-related hypotheses or new insights into the function of gd T cells.For this reason, I do not consider this work of significance to the general field of immunology or even the narrow field of gd T cell biology.

Response:
While we do extensively describe our cohort, we also conduct in-depth quantitative analysis of the γδ T cell phenotype, activation (Fig. 1, 2 and 4) and TCR repertoire (Fig. 5, 6 and 7) in CVID.Additionally, we perform functional analysis on these cells upon ex vivo stimulation (Fig. 1g).
We believe our study does lead to new and valuable insights into the function of γδ T cells.Notably, we reveal that γδ T cells can function independently of humoral immunity and remain unaffected by insufficient NF-κB signalinga concept not previously explored in human immunity.Furthermore, our research yields significant findings that generate disease-related hypotheses in the context of CVID.To date, studies on γδ T cells and CMV in humans have predominately focused on transplant or neonatal settings.Importantly, our study marks the first evidence of Vδ1 + γδ T cells activation and expansion in response to persistent CMV viremia in CVID, thereby expanding our understanding beyond transplant or neonatal settings.
We also extend these insights into clinically relevant hypotheses, in our discussion (page 13, line 335-338) we highlight that the intact functionality of γδ T cell in the context of CVID positions them as ideal candidates for future therapeutic interventions (such as γδ T cell adoptive transfer or γδ T cell targeted vaccination strategies).Given the severe and potentially fatal outcomes we observed for CVID patients with CMV disease, in our discussion we also emphasize the need for routine CMV screening and the development of targeted therapies for this at-risk patient cohort (page 13, line 320-329).
With these novel findings and insights, we are confident that our study will attract a broad readership interested in not only in γδ T cell biology, but also in virology, immunodeficiency, and clinical immunology.
Fig 2? 4. On page 17 the bioproject numbers is not indicated.5.In Fig 1B the authors use log-scale for γδ T cell frequencies.This is a bit confusing.Do the absolute numbers of Vδ1 T cells change in CVID? 6.For a number of panels within Figures the number of samples/individuals are not clearly visible.It would be important to always provide exact numbers of samples studied.Reviewer #2 (Remarks to the Author): the authors use log-scale for γδ T cell frequencies.This is a bit confusing.Do the absolute numbers of Vδ1 T cells change in CVID?Response: of panels within Figures the number of samples/individuals are not clearly visible.It would be important to always provide exact numbers of samples studied.